Ap conjugated rabbit anti goat igg antibody

To evaluate the potential effect of FHR1 on the FH-mediated regulation of the C3bBb, C3 convertase assay was performed as previously described (24 (link)). In brief, at first 5μg/ml C3b (Complement Tech) were immobilized in the microtiter plates. After blocking with 3% milk in 0.02%TBST for 2 hours at 25°C, 4μg/ml factor B (Millipore), 8μg/ml properdin (Millipore), 0.2μg/ml factor D (Millipore), 20μg/ml BSA (Sigma-Aldrich), altogether with 100nM factor H (Complement Tech) and serially diluted FHR1*A or FHR1*B (300nM-500nM) were added. After incubation, anti-factor B goat polyclonal antibody (Sigma-Aldrich) was added for incubation, followed by adding AP-conjugated rabbit anti-goat IgG antibody (Sigma-Aldrich). Finally, after incubated with alkaline phosphatase chromogenic substrate, the optical density was read at 405 nm.

SDS-PAGE and Western blot analyses were performed on purified (protein A) rCMG2-Fc variants. Protein was denatured and reduced by treating samples at 95°C for 5 min with 5% (v/v) of 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). For nonreducing SDS-PAGE, samples were denatured by heat treatment at 95°C for 5 min. Samples were loaded to precast 4–20% SDS-Tris HCl polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA), running at 200 V for 35 min. For SDS-PAGE, the gel was washed three times with water and stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Hercules, CA). For Western blot analysis, samples were transferred to a nitrocellulose membrane by electrophoretic transfer using the iBlot Gel Transfer Device (ThermoFisher, Waltham, MA). For Western blot detecting the CMG2 domain, the membrane was probed with a goat anti-CMG2 polyclonal antibody (ThermoFisher, Waltham, MA) at a concentration of 0.3 μg/ml, followed by incubation of a polyclonal AP-conjugated rabbit anti-goat IgG antibody (Sigma-Aldrich, St. Louis, MO) at 1:10,000 dilution. For Western blot detecting the Fc domain, the membrane was incubated with a polyclonal AP-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL) at 1:3,000 dilution. The blots were developed using SIGMAFAST BCIP/NBT (Sigma-Aldrich, St. Louis, MO) according to the product instruction.